1. Field of the Invention
The invention relates to novel N-substituted indole-3-glyoxylamides, processes for their preparation and pharmaceutical uses. The compounds have antiasthmatic, antiallergic and immunosuppressant/immunomodulating properties.
2. Background Information
Indole-3-glyoxylamides have various uses as pharmaco-dynamically active compounds and as synthesis components in the pharmaceutical chemistry.
The Patent Application NL 6502481 describes compounds which have an antiinflammatory and antipyretic profile of action and analgesic activity.
The British Patent GB 1 028 812 mentions derivatives of indolyl-3-glyoxylic acid and its amides as compounds having analgesic, anticonvulsant and xcex2-adrenergic activity.
G. Domschke et al. (Ber. 94, 2353 (1961)) describe 3-indolylglyoxylamides which are not characterized pharmacologically.
E. Walton et al. in J. Med. Chem. 11, 1252 (1968) report on indolyl-3-glyoxylic acid derivatives which have an inhibitory activity on glycerophosphate dehydrogenase and lactate dehydrogenase.
Euoropean Patent Specification EP 0 675 110 A1 describes 1H-indole-3-glyoxylamides which are profiled as sPLA2 inhibitors and are used in the treatment of septic shock, in pancreatitis, and in the treatment of allergic rhinitis and rheumatoid arthritis.
The aim of the present invention is to make available novel compounds from the indolyl-3-glyoxylic acid series, which have antiasthmatic and immunomodulating action.
The chemical processes for the preparation of these compounds and pharmaceutical processes for the conversion of the novel compounds into medicaments and heir preparation forms are furthermore described.
The subject matter of the invention comprises compounds of the general formula I, 
where the radicals R, R1, R2, R3, R4 and Z have the following meaning:
R=hydrogen, (C1-C6)-alkyl, where the alkyl group can be mono- or polysubstituted by the phenyl ring.
This phenyl ring, for its part, can be mono- or polysubstituted by halogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, by carboxyl groups, carboxyl groups esterified with (C1-C6)-alkanols, trifluoromethyl groups, hydroxyl groups, methoxy groups, ethoxy groups, benzyloxy groups and by a benzyl group which is mono- or polysubstituted in the phenyl moiety by (C1-C6)-alkyl groups halogen atoms or trifluoromethyl groups.
R1 can be a phenyl ring which is mono- or polysubstituted by (C1-C6)-alkyl, (C1-C6)-alkoxy, hydroxyl, benzyloxy, nitro, amino, (C1-C6)-alkylamino, (C1-C6)-alkoxy-carbonylamino and by a carboxyl group or a carboxyl group esterified by (C1-C6)-alkanols, or is a pyridine structure of the formula II 
xe2x80x83where the pyridine structure is alternatively bonded to the ring carbon atoms 2, 3 and 4 and can be substituted by the substitutents R5 and R6. The radicals R5 and R6 can be identical or different and have the meaning (C1-C6)-alkyl, and also the meaning (C3-C7)-cycloalkyl, (C1-C6)-alkoxy, nitro, amino, hydroxyl, halogen and trifluoromethyl and are furthermore the ethoxycarbonylamino radical and the group carboxyalkyloxy in which the alkyl group can have 1-4 C atoms.
R1 can furthermore be a 2- or 4-pyrimidinylheterocycle or a pyridylmethyl radical in which CH2 can be in the 2-, 3-, 4-position where the 2-pyrimidinyl ring can be mono- or polysubstituted by the methyl group, furthermore are the 2-, 3- and 4-quinolyl structure substituted by (C1-C6)-alkyl, halogen, the nitro group, the amino group and the (C1-C6)-alkylamino radical, or are a 2-, 3- and 4-quinolylmethyl group, where the ring carbons of the pyridylmethyl and quinolylmethyl radical can be substituted by (C1-C6)-alkyl, (C1-C6)-alkoxy, nitro, amino and (C1-C6)-alkoxy-carbonylamino.
R1 for the case where R is hydrogen or the benzyl group, can furthermore be the acid radical of a natural or unnatural amino acid, e.g. the xcex1-glycyl, the xcex1-sarcosyl, the xcex1-alanyl, the xcex1-leucyl, the xcex1-isoleucyl, the xcex1-seryl, the xcex1-phenylalanyl, the xcex1-histidyl, the xcex1-prolyl, the xcex1-arginyl, the xcex1-lysyl, the xcex1-asparagyl and the xcex1-glutamyl radical, where the amino groups of the respective amino acids can be present in unprotected or protected form. Possible protective groups for the amino function are the carbobenzoxy radical (Z radical) and the tert-butoxycarbonyl radical (BOC radical) and also the acetyl group. In the case of the asparagyl and glutamyl radical claimed for R1, the second, nonbonded carboxyl group is present as a free carboxyl group or in the form of an ester with C1-C6-alkanols, e.g. as the methyl, ethyl or as the tert-butyl ester. R1 can furthermore be the allylaminocarbonyl-2-methylprop-1-yl group. R and R1, together with the nitrogen atom to which they are bonded, can furthermore form a piperazine ring of the formula III or a homopiperazine ring if R1 is an aminoalkylene group in which 
R7 is an alkyl radical, a phenyl ring which can be mono- or polysubstituted by (C1-C6)-alkyl, (C1-C6)-alkoxy, halogen, the nitro group, the amino function, by (C1-C6)-alkylamino, the benzhydryl group and the bis-p-fluorobenzylhydryl group.
R2 can be hydrogen or the (C1-C6)-alkyl group, where the alkyl group can be mono- or polysubstituted by halogen and phenyl which for its part can be mono- or polysubstituted by halogen, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, carboxyl groups, carboxyl groups esterified with (C1-C6)-alkanols, trifluoromethyl groups, hydroxyl groups, methoxy groups, ethoxy groups or benzyloxy groups. The (C1-C6)-alkyl group counting as R2 can furthermore be substituted by the 2-quinolyl group and the 2-, 3- and 4-pyridyl structure, which in each case can both be mono- or polysubstituted by halogen, (C1-C4)-alkyl groups or (C1-C4)-alkoxy groups. R2 is furthermore the aroyl radical, where the aroyl moiety on which this radical is based is the phenyl ring which can be mono- or polysubstituted by halogen (C1-C6)-alkyl, (C3-C7)-cycloalkyl, carboxyl groups, carboxyl groups esterified by (C1-C6)-alkanols, trifluoromethyl groups, hydroxyl groups, methoxy groups, ethoxy groups or benzyloxy groups.
R3 and R4 can be identical or different and are hydrogen, hydroxyl, (C1-C6)-alkyl, (C3-C7)-cycloalkyl, (C1-C6)-alkanoyl, (C1-C6)-alkoxy, halogen and benzyloxy. R3 and R4 can furthermore be the nitro group, the amino group, the (C1-C4)-mono- or dialkyl-substituted amino group, and the (C1-C3)-alkoxycarbonylamino function or (C1-C3)-alkcoxycarbonylamino-(C1-C3)-alkyl function.
Z is O or S
The designation alkyl, alkanol, alkoxy or alkylamino group for the radicals R, R1, R2, R3, R4, R5, R6 and R7 is normally to be understood as meaning xe2x80x9cstraight-chainxe2x80x9d and xe2x80x9cbranchedxe2x80x9d alkyl groups, where xe2x80x9cstraight-chain alkyl groupsxe2x80x9d can be, for example, radicals such as methyl, ethyl, n-propyl, n-butyl, n-pentyl and n-hexyl and xe2x80x9cbranched alkyl groupsxe2x80x9d designate, for example, radicals such as isopropyl or tert-butyl. xe2x80x9cCycloalkylxe2x80x9d is to be understood as meaning radicals such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
The designation xe2x80x9chalogenxe2x80x9d represents fluorine, chlorine, bromine or iodine. The designation xe2x80x9calkoxy groupxe2x80x9d represents radicals such as, for example, methoxy, ethoxy, propoxy, butoxy, isopropoxy, isobutoxy or pentoxy.
The compounds according to the invention can also be present as acid addition salts, for example as salts of mineral acids, such as, for example, hydrochloric acid, sulfuric acid, phosphoric acid, salts of organic acids, such as, for example, acetic acid, lactic acid, malonic acid, maleic acid, fumaric acid, gluconic acid, glucuronic acid, citric acid, embonic acid, methanesulfonic acid, trifluoroacetic acid and succinic acid.
Both the compounds of the formula I and their salts are biologically active. The compounds of the formula 1 can be administered in free form or as salts with a physiologically tolerable acid.
Administration can be carried out orally, parenterally, intravenously, transdermally or by inhalation.
The invention furthermore relates to pharmaceutical preparations containing at least one compound of the formula I or its salt with physiologically tolerable inorganic or organic acids and, if appropriate, pharmaceutically utilizable excipients and/or diluents or auxiliaries.
Suitable administration forms are, for example, tablets, coated tablets, capsules, solutions or ampoules, suppositories, patches, powder preparations which can be inhaled, suspensions, creams and ointments.
The compounds according to the invention have a good antiasthmatic, antiallergic and immunosuppressant/immunomodulating action, for example in transplantations and diseases such as psoriasis, rheumatoid disorders and chronic polyarthritis, in the following pharmacological models:
Inhibition of the xe2x80x9cLate Phasexe2x80x9d Eosinophilia in the BAL 24 Hours After Allergen Challenge in Guinea Pigs
Male guinea pigs (200-250 g, Dunkin Hartley Shoe) were actively sensitized subcutaneously with ovalbumin (10 xcexcg of ovalbumin+1 mg of Al(OH)3) and boosted 2 weeks later. One week after boosting with ovalbumin, the animals were exposed to an inhalation challenge with ovalbumin (0.5% strength solution) for 20-30 seconds. 24 hours later, the animals were killed by means of an overdose of urethane, exsanguinated and a bronchoalveolar lavage (BAL) was carried out using 2xc3x975 ml of 0.9% strength physiological saline solution.
The lavage fluid was collected and centrifuged at 400 g for 10 minutes, and the pellets were suspended in 1 ml of 0.9% strength physiological saline solution. The eosinophils were counted microscopically in a Neubauer chamber after staining by means of Becton Dickinson test kit No. 5877. This test kit contains Phloxin B as a selective stain for eosinophils. The eosinophils in the BAL was [sic] counted here for each animal and expressed as eosinophils (millions/animal). For each group the mean value and standard deviation were determined. The percentage inhibition of eosinophilia for the group treated with test substance was calculated according to the following formula:
(Axe2x88x92B)xe2x88x92(Bxe2x88x92C)/(Axe2x88x92C)xc3x97100=% inhibition
in this formula A eosinophils correspond to the untreated challenge group, B eosinophils to the treated group and C eosinophils to the unchallenged control group.
The animals were treated with a histamine H1 antagonist (azelastine; 0.01 mg/kg p.o.) 2 hours before allergen challenge to avoid death. The administration of the test substances or of the vehicle was carried out 4 hours after allergen challenge. The percentage inhibition of eosinophilia in the BAL was calculated on groups of 6-10 animals.
Assays for the Determination of Peptidylprolyl Isomerase (PPIase) Activity and Inhibition
The PPIase activity of the cyclophilins was measured enzymatically according to Fischer et al. (1984). After isomerization of the substrate by the peptidyl prolyl isomerase, this is accessible to chymotrypsin, which cleaves the chromophore p-nitroaniline. For the determination of inhibition of the PPIase activity by substance, recombinant human Cyp B was used. The interaction of Cyp B with a potential inhibitor was carried out as follows:
A certain concentration of purified Cyp B was incubated with 1 xcexcM substance for 15 min. The PPIase reaction was started by addition of the substrate solution to the reaction mixture which contains HEPES buffer, chymotrypsin and either test or control samples. Under these conditions, first-order kinetics were obtained with a constant Kobserved=K0+Kenz, where K0 is the spontaneous isomerization and Kenz is the rate of isomerization of the PPIase activity. The extinction values which correspond to the amount of the chromophore cleaved were measured using a Beckman DU 70 spectrophotometer at a constant reaction temperature of 10xc2x0 C.
The observed residual activity in the presence of various substances was compared with the cyclophilins only treated with solvent. The results were given in % residual activity. Cyclosporin A (CsA) was used as the reference compound. The inhibition of the PPIase activity was additionally checked by SDS-PAGE.
Colorimetric assay (based on the MTT test) for the non-radioactive quantification of cell proliferation and survival ability
MTT is used for the quantitative determination of cell proliferation and activation, for example, in the reaction on growth factors and cytokines such as IL-2 and IL-4 and also for the quantification of the antiproliferative or toxic effects.
The assay is based on the cleavage of yellow tetrazolium salt MTT to give purple-red formazan crystals by metabolically active cells.
The cells, cultured in a 96-hole tissue culture plate, are incubated for about 4 h with yellow MTT solution. After this incubation time, purple-red formazan salt crystals are formed. These salt crystals are insoluble in aqueous solutions, but can be dissolved by addition of solubilizer and by incubation of the plates overnight.
The dissolved formazan product is quantified spectrophotometrically using an ELISA reader. An increase in the number of living cells results in an increase in the total metabolic activity in the sample. This increase correlates directly with the amount of the purple-red formazan crystals formed, which are [sic] measured by the absorption.
The processes for the preparation of the compounds according to the invention are described in the following reaction schemes 1 and 2 and in general procedures. All compounds can be prepared as described or analogously.
The compounds of the general formula I are obtainable according to the following Scheme 1, shown for the synthesis of the compound Example 1: 
1st Stage:
The indole derivative, which can be unsubstituted or mono- or polysubstituted on C-2 or in the phenyl structure, is dissolved in a protic, dipolar aprotic or nonpolar organic solvent, such as, for example, isopropanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dioxane, toluene or methylene chloride and added dropwise to a suspension of a base in a molar or excess amount prepared in a 3-necked flask under an N2 atmosphere, such as, for example, sodium hydride, powdered potassium hydroxide, potassium tert-butoxide, dimethylaminopyridine or sodium amide in a suitable solvent. The desired alkyl, aralkyl or heteroaralkyl halide, if appropriate with addition of a catalyst, such as, for example, copper, is then added and the mixture is reacted for some time, for example 30 minutes to 12 hours, and the temperature is kept within a range from 0xc2x0 C. to 120xc2x0 C., preferably between 30xc2x0 C. to [sic] 80xc2x0 C., particularly between 50xc2x0 C. and 65xc2x0 C. After completion of the reaction, the reaction mixture is added to water, the solution is extracted, for example, with diethyl ether, dichloromethane, chloroform, methyl tert-butyl ether or tetrahydrofuran and the organic phase obtained in each case is dried using anhydrous sodium sulfate. The organic phase is concentrated in vacuo, the residue which remains is crystallized by trituration or the oily residue is purified by recrystallization, distillation or by column or flash chromatography on silica gel or alumina. The eluent used is, for example, a mixture of dichloromethane and diethyl ether in the ratio 8:2 (vol/vol) or a mixture of dichloromethane and ethanol in the ratio 9:1 (vol/vol).
2nd Stage
The N-substituted indole obtained by the abovementioned 1st stage procedure is dissolved under a nitrogen atmosphere in an aprotic or nonpolar organic solvent, such as, for example, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, dioxane, toluene, xylene, methylene chloride or chloroform and added to a solution, prepared under a nitrogen atmosphere, of a simply molar up to 60 percent excess amount of oxalyl chloride in an aprotic or nonpolar solvent, such as, for example, in diethyl ether, methyl tert-butyl ether, tetrahydrofuran, dioxane, toluene, xylene, methylene chloride or chloroform, the temperature being kept between xe2x88x925xc2x0 C. and 20xc2x0 C. The reaction solution is then heated at a temperature between 10xc2x0 C. and 130xc2x0 C., preferably between 20xc2x0 C. and 80xc2x0 C., particularly between 0xc2x0 C. and 50xc2x0 C., for a period of 30 minutes up to 5 hours and the solvent is then evaporated. The residue of the xe2x80x9cindolyl-3-glyoxylic acid chloridexe2x80x9d formed in this manner which remains is dissolved in an aprotic solvent such as, for example, tetrahydrofuran, dioxane, diethyl ether, toluene or alternatively in a dipolar aprotic solvent, such as, for example, dimethylformamide, dimethylacetamide or dimethyl sulfoxide, cooled to a temperature between 10xc2x0 C. and xe2x88x9215xc2x0 C., preferably between xe2x88x925xc2x0 C. and 0xc2x0 C., and treated in the presence of an acid scavenger with a solution of the primary or secondary amine in a diluent.
Possible diluents are the solvents used above for the dissolution of the indolyl-3-glyoxylic acid chloride. Acid scavengers used are triethylamine, pyridin, dimethylaminopyridine, basic ion exchanger, sodium carbonate, potassium carbonate, powdered potassium hydroxide and excess primary or secondary amine employed for the reaction. The reaction takes place at a temperature from 0xc2x0 C. to 120xc2x0 C., preferably at 20-80xc2x0 C., particularly between 40xc2x0 C. and 60xc2x0 C. After a reaction time of 1-3 hours and standing at room temperature for 24 hours, the hydrochloride of the acid scavenger is filtered, the filtrate is concentrated in vacuo, and the residue is recrystallized from an organic solvent or purified by column chromatography on silica gel or alumina. The eluent used is, for example, a mixture of dichloromethane and ethanol (95:5, vol/vol).